Biomedical subjects

J R Meyer-Fernandes

Publications and source records attributed to J R Meyer-Fernandes.

At least 37 records · Page 2Linked to original sources

PubMed1999

A novel role of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid as an activator of the phosphatase activity catalyzed by plasma membrane Ca2+-ATPase.

The hydrolysis of p-nitrophenyl phosphate catalyzed by the erythrocyte membrane Ca2+-ATPase is stimulated by low concentrations of the compound 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a classic inhibitor of anion transport. Enhancement of the phosphatase activity varies from 2- to 6-fold, depending on the Ca2+ and calmodulin concentrations used. Maximum stimulation of the pNPPase activity in ghosts is reached at 4-5 microM DIDS. Under the same conditions, but with ATP rather than pNPP as the substrate, the Ca2+-ATPase activity is strongly inhibited. Activation of pNPP hydrolysis by DIDS is equally effective for both ghosts and purified enzyme, and therefore is independent of its effect as an anion transport inhibitor. Binding of the activator does not change the Ca2+ dependence of the pNPPase activity. Stimulation is partially additive to the activation of the pNPPase activity elicited by calmodulin and appears to involve a strong affinity binding or covalent binding to sulfhydryl groups of the enzyme, since activation is reversed by addition of dithiothreitol but not by washing. The degree of activation of pNPP hydrolysis is greater at alkaline pH values. DIDS decreases the apparent affinity of the enzyme for pNPP whether in the presence of Ca2+ alone or Ca2+ and calmodulin or in the absence of Ca2+ (with 5 microM DIDS the observed Km shifts from 4.8 +/- 1.4 to 10.1 +/- 2.6, from 3.8 +/- 0.4 to 7.0 +/- 0.8, and from 9.3 +/- 0.7 to 15.5 +/- 1.1 mM, respectively). However, the pNPPase rate is always increased (as above, from 3.6 +/- 0.6 to 11.2 +/- 1.7, from 4.4 +/- 0.5 to 11.4 +/- 0.9, and from 2.6 +/- 0.6 to 18.6 +/- 3.9 nmol mg-1 min-1, in the presence of Ca2+ alone or Ca2+ and calmodulin or in the absence of Ca2+, respectively). ATP inhibits the pNPPase activity in the absence of Ca2+, both in the presence and in the absence of DIDS. Therefore, kinetic evidence indicates that DIDS does more than shift the enzyme to the E2 conformation. We propose that the transition from E2 to E1 is decreased and a new enzyme conformer, denoted E2*, is accumulated in the presence of DIDS.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1999

Altered tyrosine phosphorylation of ERK1 MAP kinase and other macrophage molecules caused by Leishmania amastigotes.

The involvement of tyrosine phosphorylation during macrophage infection with Leishmania amazonensis amastigotes was investigated. PTK antagonists such as genistein, herbimycin A, geldanamycin and tyrphostin 25 had no significant effect on adhesion to, or entry into, murine peritoneal macrophages, but increased parasite intracellular survival. LPS-induced tyrosine phosphorylation of target host proteins assessed by immunoprecipitation and Western blot was impaired or reversed by living amastigotes soon after 60 min-infection. Such reversion was not due to parasite-secreted molecules but was contact-dependent, as assessed by cytochalasin D treatment of macrophage monolayers prior to infection. Paraformaldehyde-fixed or sodium vanadate-treated amastigotes exerted no significant effect on overall macrophage tyrosine phosphorylation. Immunoprecipitation of proteins employing 4G10 anti-phosphotyrosine antibody followed by Western blotting revealed that tyrosine phosphorylation of 120, 85, 60, 44 and 35 kDa proteins was selectively reversed by amastigote infection. Inhibition, measured by densitometry was from about 66-100% of uninfected cells. None of these proteins was immunoprecipitated from amastigote-infected macrophage lysates but all of them except for p85 were recovered after treatment of parasites with 100 microM sodium orthovanadate prior to infection, a treatment that inhibits Leishmania amastigote protein ecto-phosphatase. The 44 kDa protein was identified as ERK1 MAP kinase (MAPK) by Western blot. Amastigote infection also decreased tyrosine phosphorylation induced by zymosan particles. Vanadate treatment of amastigotes prior to infection significantly decreased parasite intracellular survival. The action of a putative leishmanial ecto-protein phosphatase (PPase) is suggested.

Animals↗

PubMed1999

Protection against thermal denaturation by trehalose on the plasma membrane H+-ATPase from yeast. Synergetic effect between trehalose and phospholipid environment.

Yeast cells have had to develop mechanisms in order to protect themselves from chemical and physical agents of the environment to which they are exposed. One of these physical agents is thermal variation. Some yeast cells are known to accumulate high concentrations of trehalose when submitted to heat shock. In this work, we have studied the effect of trehalose on the protection against thermal inactivation of purified plasma membrane H+-ATPase from Schizosaccharomyces pombe, in the solubilized and in the reconstituted state. We observed that after 1 min of incubation at 51 degrees C in the presence of 1 M trehalose, about 50% of soluble enzyme remains active. In the same conditions, but in the absence of trehalose, the activity was completely abolished. The t0.5 for the enzyme inactivation increased from 10 to 50 s after reconstitution into asolectin liposomes. Curiously, in the presence of 1 M trehalose, the t0.5 for inactivation of the reconstituted enzyme was further increased to higher than 300 s, regardless of whether trehalose was added inside or outside the liposome. Additionally, the concentration that confers 50% for the protection by trehalose (K0.5) decreased from 0.5 M, in the solubilized state, to 0.04 M in the reconstituted state, suggesting a synergetic effect between sugar and lipids. Gel electrophoresis revealed that the pattern of H+-ATPase cleavage by trypsin changed when 1 M trehalose was present in the buffer. It is suggested that both in a soluble and in a phospholipid environment, accumulation of trehalose leads to a more heat-stable conformation of the enzyme, probably an E2-like form.

Adenosine Triphosphatases↗

PubMed1999

Ecto-phosphatase activities on the cell surface of the amastigote forms of Trypanosoma cruzi.

Live Trypanosoma cruzi amastigotes hydrolyzed p-nitrophenylphosphate (PNPP), phospho-amino-acids and 32P-casein under physiologically appropriate conditions. PNPP was hydrolysed at a rate of 80 nmol.mg-1.h-1 in the presence of 5 mM MgCl2, pH 7.2 at 30 degrees C. In the absence of Mg2+ the activity was reduced 40% and we call this basal activity. At saturating concentration of PNPP, half-maximal PNPP hydrolysis was obtained with 0.22 mM MgCl2. Ca2+ had no effect on the basal activity, could not substitute Mg2+ as an activator and in contrast inhibited the PNPP hydrolysis stimulated by Mg2+ (I50 = 0.43 mM). In the absence of Mg2+ (basal activity) the stimulating half concentration (S0.5) for PNPP was 1.57 mM, while at saturating MgCl2 concentrations the corresponding S0.5 for PNPP for Mg(2+)-stimulated phosphatase activity (difference between total minus basal phosphatase activity) was 0.99 mM. The Mg-dependent PNPP hydrolysis was strongly inhibited by sodium fluoride (NaF), vanadate and Zn2+ but not by tartrate and levamizole. The Mg-independent basal phosphatase activity was insensitive to tartrate, levamizole as well NaF and less inhibited by vanadate and Zn2+. Intact amastigotes were also able to hydrolyse phosphoserine, phosphothreonine and phosphotyrosine but only the phosphotyrosine hydrolysis was stimulated by MgCl2 and inhibited by CaCl2 and phosphotyrosine was a competitive inhibitor of the PNPP hydrolysis stimulated by Mg2+. The cells were also able to hydrolyse 32P-casein phosphorylated on serine and threonine residues but only in the presence of MgCl2. These results indicate that in the amastigote form of T. cruzi there are at least two ectophosphatase activities, one of which is Mg2+ dependent and can dephosphorylate phospho-amino acids and phosphoproteins under physiological conditions.

4-Nitrophenylphosphatase↗

PubMed1998

A novel ecto-phosphatase activity of Herpetomonas muscarum muscarum inhibited by platelet-activating factor.

In the present work ecto-phosphatase activity in Herpetomonas muscarum muscarum has been characterized using live parasites. This enzyme hydrolyzed p-nitrophenylphosphate at a rate of 4.27 nmol Pi/mg of protein.min. A pH curve was generated, in which these intact flagellates showed the highest phosphatase activity at pH 6.5. Classical inhibitors for acid phosphatase, such as sodium orthovanadate, sodium tartrate, and ammonium molybdate, were used in the experiments and showed different patterns of inhibition. Lithium fluoride, aluminum chloride, and fluoroaluminate complexes were also tested. Although lithium fluoride and fluoroaluminate complexes were capable of inhibiting the phosphatase activity, aluminum chloride stimulated this enzyme. Cytochemical analysis showed the localization of this enzyme on the parasite surface. This ecto-phosphatase activity was also significantly diminished when the parasites were treated with 10(-6) M platelet-activating factor (PAF), a potent phospholipid mediator that promoted cellular differentiation in this parasite.

4-Nitrophenylphosphatase↗

PubMed1998

Stabilization against thermal inactivation promoted by sugars on enzyme structure and function: why is trehalose more effective than other sugars?

Trehalose has been described to act as the best stabilizer of structure and function of several macromolecules. Although other sugars also stabilize macromolecules, none of them are as effective as trehalose. The extraordinary effect of trehalose has been attributed to several of its properties such as making hydrogen bonds with membranes or the ability to modify the solvation layer of proteins. However, the explanations always result in a question: Why is trehalose more effective than other sugars? Here, we show that trehalose has a larger hydrated volume than other related sugars. According to our results, trehalose occupies at least 2.5 times larger volume than sucrose, maltose, glucose, and fructose. We correlate this property with the ability to protect the structure and function of enzymes against thermal inactivation. When the concentrations of all sugars were corrected by the percentage of the occupied volume, they presented the same effectiveness. Our results suggest that because of this larger hydrated volume, trehalose can substitute more water molecules in the solution, and this property is very close to its effectiveness. Finally, these data drive us to conclude that the higher size exclusion effect is responsible for the difference in efficiency of protection against thermal inactivation of enzymes.

Enzyme Activation↗

PubMed1998

Ecto-protein tyrosine phosphatase activity in Trypanosoma cruzi infective stages.

Live T. cruzi trypomastigotes and amastigotes possess ecto-protein tyrosine phosphatase activity as indicated by the ability of intact cells to catalyze dephosphorylation of tyrosine phosphorylated myelin basic protein, [32P]TyrRaytide, phosphotyrosine, or the phosphotyrosine analog p-nitrophenylphosphate (p-NPP). The dephosphorylation of myelin basic protein (MBP) and p-NPP was inhibited by sodium o-vanadate, zinc chloride and NaF, while dephosphorylation of [32P]TyrRaytide was insensitive to zinc chloride but sensitive to o-vanadate and NaF. In contrast, live cells were not able to dephosphorylate serine or threonine phosphorylated peptides ([32P]Kemptide) or proteins ([32P]RCM-lysozyme and [32P]MBP).

Animals↗

PubMed1998

Ouabain-insensitive Na(+)-ATPase activity of Malpighian tubules from Rhodnius prolixus.

In the present paper, we show the existence of a furosemide-sensitive Na(+)-stimulated, Mg(2+)-dependent ATPase activity in cell lysates of Malpighian tubular cells from Rhodnius prolixus, which could be the biochemical expression of the Na(+)-pump. The main characteristics of this activity are: (1) K0.5 for Na+ = 1.49 +/- 0.18 mM, (2) Vmax = 2.8 +/- 0.1 nmol inorganic orthophosphate (Pi).mg prot-1.min-1, (3) it is fully abolished by 2 mM furosemide, (4)it is insensitive to ouabain concentrations up to 10(-2) M, (5) it is sensitive to the presence of vanadate in the incubation medium indicating it to be a P-type ATPase, and (6) it is stimulated by nanomolar concentrations of Ca2+ in the incubation medium.

Adenosine Triphosphatases↗

PubMed1997

Carbohydrate protection of enzyme structure and function against guanidinium chloride treatment depends on the nature of carbohydrate and enzyme.

Baker's yeast cells accumulate osmolytes as a response to several stress conditions such as high-temperature and low-temperature shifts, dehydration, or osmotic stress. One of the major osmolytes that accumulates is trehalose, which plays an essential role affecting the survival of yeast at the time of stress. In this report, we show that trehalose efficiently protects the function and the structure of two yeast cytosolic enzymes against chemical denaturation by guanidinium chloride. Other sugars tested also protected yeast pyrophosphatase and glucose-6-phosphate dehydrogenase structure against guanidinium chloride effects, but were not as efficient at protecting enzyme activity. The thermostable pyrophosphatase from Bacillus stearothermophilus was also protected by several sugars against the chaotropic properties of guanidinium chloride, but was only protected by trehalose against functional inactivation. The function of the membrane-embedded H+-ATPase from yeast could not be protected by any of the tested sugars, although all of the sugars protected its structure from guanidinium-chloride-induced unfolding. The results presented in this study suggest that several sugars are able to prevent protein unfolding induced by a chaotropic compound. However, prevention of functional inactivation depends on the nature of the sugar. Trehalose was the most efficient, being able to protect many cytosolic enzymes against guanidinium chloride. The efficiency of protection also depended on the nature of the protein tested. This might explain why trehalose is one of the osmolytes accumulated in yeast and also why it is not the only osmolyte to accumulate.

Carbohydrates↗

PubMed1997

Mg-dependent ecto-ATPase activity in Leishmania tropica.

ATPase activity has been located on the external surface of Leishmania tropica. Since Leishmania is known to have an ecto-acid phosphatase, in order to discard the possibility that the ATP hydrolysis observed was due to the acid phosphatase activity, the effect of pH in both activities was examined. In the pH range from 6.8 to 8.4, in which the cells were viable, the phosphatase activity decreased, while the ecto-ATPase activity increased. To confirm that the observed ATP hydrolysis was promoted by neither phosphatase nor 5'-nucleotidase activities, a few inhibitors for these enzymes were tested. Vanadate and NaF strongly inhibited the phosphatase activity; however, no effect was observed on ATPase activity. Neither levamizole nor tetramizole, two specific inhibitors of alkaline phosphatases, inhibited this activity. The lack of response to ammonium molybdate indicated that 5'-nucleotidase did not contribute to the ATP hydrolysis. Also, the lack of inhibition of the ATP hydrolysis by high concentrations of ADP at nonsaturating concentrations of ATP discarded the possibility of any ATP diphosphohydrolase activity. The ATPase here described was stimulated by MgCl2 but not by CaCl2. In the absence of divalent metal, a low level of ATP hydrolysis was observed, and CaCl2 varying from 0.1 to 10 mM did not increase the ATPase activity. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.29 +/- 0.02 mM MgCl2. The apparent K(m) for Mg-ATP2- was 0.13 +/- 0.01 mM and free Mg2+ did not increase the ATPase activity. ATP was the best substrate for this enzyme. Other nucleotides such as ITP, CTP, GTP, UTP, and ADP produced lower reaction rates. To confirm that this Mg-dependent ATPase was an ecto-ATPase, an impermeant inhibitor, 4,4'-diisothiocyanostylbene-2,2'-disulfonic acid was used. This amino/sulfhydryl-reactive reagent did inhibit the Mg-ecto-ATPase activity in a dose-dependent manner (I0.5 = 27.5 +/- 1.8 microM).

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1997

Ultrastructural and biochemical analysis of the relationship of Crithidia deanei with its endosymbiont.

Some protozoa of the Trypanosomatidae family harbor in their cytoplasm bacterial endosymbionts that provide essential nutrients to and induce morphological alterations in the protozoa. In the present study, a close association between endosymbionts and glycosomes, a peroxisome-like organelle where most of the enzymes of the glycolytic pathway are compartmentalized, was identified by conventional transmission electron microscopy in Crithidia deanei. Such an association was further supported by the cytochemical localization of catalase in the glycosome and also confirmed by 3-D reconstruction of the protozoan. The enzymes cytochrome oxidase and succinate dehydrogenase were detected by ultrastructural cytochemistry. A positive reaction was observed in the protozoan mitochondrion but not in the endosymbiont envelope. Enzymatic assays for succinate cytochrome c reductase reinforced these results, as a low enzymatic activity was detected in an endosymbiont-enriched fraction, while high activity was observed in a purified protozoan mitochondrion fraction. We also demonstrated that a purified symbiont fraction was able to hydrolyze ATP. This activity was Mg+2 dependent, since it was highly stimulated by the presence of physiological concentrations of this ion. Taken together, these observations suggest that no electron transporting system is active in the symbionts of Crithidia deanei and that they might obtain energetic molecules derivated from the protozoan glycosomes.

Adenosine Triphosphatases↗

PubMed1996

Leishmania-induced tyrosine phosphorylation in the host macrophage and its implication to infection.

Tyrosine phosphorylation is an important mechanism of cell regulation and has been recently implicated in defense strategies against a variety of pathogens. We have investigated the involvement of protein tyrosine kinase activity in the Leishmania attachment, invasion and survival within macrophages, as well as promastigote ability to trigger tyrosine phosphorylation, which could contribute to leishmanicidal activity. Treatment of murine macrophage monolayers with genistein, herbimycin A, tyrphostin 25 or staurosporine prior to infection decreased parasite invasion in a dose-dependent manner. Contrary, addition of sodium orthovanadate, a protein tyrosine phosphatase inhibitor, phosphotyrosine and p-nitrophenyl phosphate to the interaction medium, significantly increased parasite binding and internalization, whereas phosphoserine and phosphothreonine had no effect. The phosphatase activity of intact promastigotes was greater than that of macrophages. Western blot analysis revealed tyrosine-phosphorylated bands from 198 to 28 kDa following macrophage challenge with promastigotes. Uninfected macrophages displayed no detectable tyrosine phosphorylated proteins, possibly indicating an inducible process, while in parasites it was constitutive, as seen by the presence of 42, 40 and 35 kDa phosphoproteins on the Leishmania lysates. Immunofluorescence and immunogold detection of phosphotyrosine residues in some promastigote-macrophage attachment areas, but not in the vicinity of ingested parasites, suggest that Leishmania-induced tyrosine phosphorylation is an early, local and short-lived event. Genistein treatment of Leishmania-infected cells significantly enhanced the parasite burden. This antagonist also diminished nitric oxide production in resting and interferon gamma/lipopolysaccharide-activated infected macrophages, which may account for the increased parasite survival. We propose that protein tyrosine kinase-linked pathways regulate the Leishmania promastigote invasion and the macrophage microbicidal activity.

Animals↗

PubMed1995

Reactions involving carbamyl phosphate in the presence of precipitated calcium phosphate with formation of pyrophosphate: a model for primitive energy-conservation pathways.

The formation of carbamyl phosphate (CAP) in dilute solutions of cyanate (NCO-) and orthophosphate (Pi) was measured both in the absence and in the presence of a precipitated matrix of calcium phosphate (Pi.Ca). The second-order rate constant and the free energy of CAP synthesis were not modified by the presence of the solid matrix, indicating that synthesis occurs in the homogeneous Pi-containing solution. The elimination reaction of CAP to form NCO- and Pi followed first-order kinetics and the rate constant was the same whether or not calcium phosphate was present. Elimination was not complete, and the steady level of remaining CAP was that expected from the free energy of synthesis. The formation of pyrophosphate (PPi) was detected in CAP-containing medium only in the presence of calcium, showing a close correlation with the amount of precipitated Pi.Ca. Phosphorolysis of CAP followed a sigmoidal time course, compatible with adsorption of CAP to the solid matrix as a prelude to transphosphorylation. Addition of 5'-AMP and of short linear polyphosphates inhibited phosphorolysis of CAP. It is proposed that the presence of a solid phosphate matrix and the relative concentrations of cyano compounds, as well as those of nucleotides and inorganic polyphosphates, could have played a crucial role in the conservation of chemical energy of CAP and in its use in prebiotic phosphorylation reactions.

Adenosine Monophosphate↗

PubMed1995

Leishmanial protein kinase C modulates host cell infection via secreted acid phosphatase.

To study the role of parasite protein kinase C (PKC) activity in the uptake of Leishmania amazonensis by mononuclear phagocytes we treated the parasites with 12-O-tetradecanoyl phorbol-13-acetate (TPA) and/or sphingosine, before interaction assays. Promastigotes of Leishmania amazonensis were incubated with 20 ng/ml TPA and/or 50 ng/ml sphingosine before the interaction with murine peritoneal macrophages. The short treatment enhanced about 200% the parasite association with the host cells, whereas the sphingosine treatment reduced about 50% the promastigote binding, as did the prolonged TPA treatment. The binding of cells treated with both drugs was not significantly altered. Biochemical and cytochemical data indicate that the protein kinase C agonists TPA and sphingosine, respectively, increased and decreased acid phosphatase (AcP) activity. The addition of sodium tartrate, a secreted AcP inhibitor, suppressed the TPA enhancing effects, but did not affect the basal parasite binding observed in control cells. The supernatants of TPA-treated L. amazonensis promastigotes increased the parasite association by about the same extent as the TPA treatment, and this effect was also abolished by tartrate. Although TPA did not enhance the association of L. major, a species that does not secrete AcP, the supernatants of TPA-treated L. amazonensis increased it in a tartrate-sensitive manner. The results suggest that Leishmania amazonensis PKC activity may modulate its interaction with macrophages via secreted AcP.

Acid Phosphatase↗

PubMed1994

Ca(2+)-dependent permeabilization of the inner mitochondrial membrane by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS).

We have recently shown that the permeabilization of the inner mitochondrial membrane by Ca2+ plus prooxidants is associated with oxidation of protein thiols forming cross-linked protein aggregates (Fagian, M.M., Pereira-da-Silva, L., Martins, I.S. and Vercesi, A.E. (1990) J. Biol. Chem. 265, 19955-19960). In this study we show that the incubation of rat liver mitochondria in the presence of the thiol reagent 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and Ca2+ caused production of membrane protein aggregates, mitochondrial swelling, disruption of membrane potential and Ca2+ release. The presence of DTT prevented but did not reverse the elimination of delta psi induced by DIDS. EGTA prevented delta psi elimination and decreased the amount of protein aggregates, suggesting that the binding of Ca2+ to some membrane protein may expose buried thiols to react with DIDS. Reversal of collapsed delta psi by EGTA indicates that DIDS-induced protein aggregates require the presence of Ca2+ for significant membrane permeabilization. Cyclosporin A prevented mitochondrial swelling, suggesting that DIDS-induced membrane protein polymerization mimics the condition designated as Ca(2+)-induced permeabilization transition of mitochondria. The lack of oxidation of pyridine nucleotides or significant lipid peroxidation by DIDS supports the notion that membrane permeabilization by this compound is mediated by its interaction with membrane proteins.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1992

A (Ca(2+)-Mg2+)ATPase from Schistosoma mansoni is coupled to an active transport of calcium.

The (Ca(2+)-Mg2+)ATPase activity in microsomes of Schistosoma mansoni is fully inhibited by vanadate (I50 = 2.5 microM). 45Ca2+ is accumulated within microsomal vesicles in an ATP-dependent process that is enhanced 5-fold in the presence of 40 mM phosphate. Accumulated 45Ca2+ is rapidly released by 5 microM of the Ca2+ ionophore A23187 (t1/2 less than or equal to 6 s). (Ca(2+)-Mg2+)ATPase activity and Ca2+ uptake share the same subcellular distribution pattern and similar Ca2+ sensitivities (K0.5 = 0.39 microM and 0.15 microM, respectively). The substrate selectivity is high for both ATPase activity and Ca2+ transport. These results indicate the presence of an active transport of Ca2+ coupled to the (Ca(2+)-Mg2+)ATPase activity previously described in this parasite. A plasma membrane localization and physiological role in calcium homeostasis are suggested.

Animals↗

PubMed1991

Reversible inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid of the plasma membrane (Ca(2+)+Mg2+)ATPase from kidney proximal tubules.

Calcium accumulation by purified vesicles derived from basolateral membranes of kidney proximal tubules was reversibly inhibited by micromolar concentrations of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of anion transport. The inhibitory effect of this compound on Ca2+ uptake cannot be attributed solely to the inhibition of anion transport: (Ca(2+)+Mg2+)ATPase and ATP-dependent Ca2+ transport, respectively. The rate constant of EGTA-induced Ca2+ efflux from preloaded vesicles was not affected by DIDS, indicating that this compound does not increase the permeability of the membrane vesicles to Ca2+. In the presence of DIDS, the effects of the physiological ligands Ca2+, Mg2+, and ATP on (Ca(2+)+Mg2+)ATPase activity were modified. The Ca2+ concentration that inhibited (Ca(2+)+Mg2+)ATPase activity in the low-affinity range decreased from 91 to 40 microM, but DIDS had no effect on the Km for Ca2+ in the high-affinity, stimulatory range. Free Mg2+ activated (Ca(2+)+Mg2+)ATPase activity at a low Ca2+ concentration, and DIDS impaired this stimulation in a noncompetitive fashion. The inhibition by DIDS was eliminated when the free ATP concentration of the medium was raised from 0.3 to 8 mM, possibly due to an increase in the turnover of the enzyme caused by free ATP accelerating the E2----E1 transition, and leading to a decrease in the proportion of E2 forms under steady-state conditions. Alkaline pH totally abolished the inhibition of the (Ca(2+)+Mg2+)ATPase activity by DIDS, with a half-maximal effect at pH 8.3.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1991

ATP in equilibrium with 32Pi exchange catalyzed by plasma membrane Ca(2+)-ATPase from kidney proximal tubules.

The Ca(2+)-stimulated adenosine 5'-triphosphate-orthophosphate (ATP in equilibrium with 32Pi) exchange reaction was studied using a vesicular preparation derived from plasma membrane of kidney proximal tubules. With native inside-out vesicles, ATP in equilibrium with 32Pi was stimulated by micromolar Ca2+ concentrations. Treatment of the vesicles with the Ca2+ ionophore A23187 that abolished Ca2+ accumulation, strongly inhibited ATP in equilibrium with 32Pi. When Ca(2+)-ATPase was solubilized with the nonionic detergent octaethylene glycol mono n-dodecyl ether, maximal activation of ATP in equilibrium with 32Pi required millimolar Ca2+ concentrations. These Ca2+ concentrations inhibited ATP hydrolysis. ATP in equilibrium with 32Pi exhibited a Michaelian dependence on Pi and Mg2+, was stimulated by ATP, and depended on the ATP/ADP ratio. ATP in equilibrium with 32Pi was modified by the osmolytes urea, trimethylamine-N-oxide, and sucrose, which are representative of the methylamines and polyols that normally accumulate in renal tissue. These compounds did not modify the apparent affinity for Pi; they affected the response to ADP in the same fashion as the overall rate of ATP in equilibrium 32Pi, and their effects depended on medium pH. These data show that the Ca(2+)-ATPase from plasma membrane kidney proximal tubules can operate simultaneously in forward and backward directions. They also show that ATP in equilibrium with 32Pi is modulated by the ligands Ca2+, ATP, ADP, Pi, Mg2+, and H+, and by organic solutes found in renal tissue.

Adenosine Diphosphate↗